Carboxamide-substituted xanthene dyes

ABSTRACT

Carboxamide-substituted xanthene dyes, reactive dyes, and the use of such dyes as a labeling reagent are disclosed. Specifically, a carboxamide-substituted dye of the formula (I) 
     
       
         
         
             
             
         
       
     
     is disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention disclosesxanthene-based, carboxamide-substituted dyes of the general formula (I)as well as the preparation, activation, and use of said dyes.

The dyes according to formula (I) have very good spectral properties,such as the position of the absorbance and emission bands, highextinction coefficient, high fluorescent quantum yields, andphotostability. The disadvantage of lactone or lactam formation, whichis associated with the presence of carboxylic acid at the 2^(nd)position of the “lower” aromatic ring, is prevented by the conversion ofsaid carboxylic acid group into a secondary amide.

The dyes of this invention possess considerable advantage over theirpreviously described xanthene dyes. In particular, their fluorescenceyields are typically higher than those of other dyes having compatiblespectra, including fluorescein, Cy-2, tetramethylrhodamine, Cy-3, andTexas Red. In addition, the dyes of this invention exhibit enhancedresistance to quenching upon protein conjugation, and protein conjugateswith the dyes of the invention typically possess substantially higherfluorescence yields than that achieved with most commercially availablefluorescent dyes, including AlexaFluor dyes. Also, the dyes of thisinvention are substantially more water-soluble than dyes without anamido-sulfoalkyl group.

2. Description of the Background

Organic fluorescent compounds, also known as dyes, are widely used assensitive detection reagents in biological systems. Xanthene-type dyesare among the most frequently organic fluorescent compounds used asdetection reagents due to their very good spectral properties andphotostability. The dyes having a very high fluorescence quantum yieldare especially important since the fluorescence enables the labeledanalyte to be detected at very high sensitivity.

Many xanthene-type dyes possess a carbonyl group at the o-position ofthe “lower” aromatic ring, which causes the formation of a colorlesslactone under some physiological conditions. The lactone is colorlessand non-fluorescent. Thus, the labeled analyte cannot be detected bymeans of fluorescent spectroscopy.

The traditional way to covalently attach a fluorescent dye to abiomolecule is through a reaction of a primary amine group of thebiomolecule with an activated ester, for example dyes of NHS ester. Oneway of rendering the formation of non-fluorescent lactone is to use thecarboxylic group o-position of the “lower” aromatic ring for covalentattachment of a fluorescent dye to the biomolecule. However, thisreaction produces a primary amide, which immediately rearranges into alactam according to Scheme 1.

The lactam is colorless and non-fluorescent under physiologicallyrelevant conditions. Thus, the labeled analyte cannot be detected bymeans of fluorescent spectroscopy.

WO 02/055512 and US 2006/0154251 A disclose the preparation of amidederivatives of fluorescein and rhodamine dyes, which comprise theconversion of carboxylic acids into activated esters followed byreaction of said activated esters with a secondary amine under refluxconditions to form secondary amides. Even though the disclosed secondaryamides do not form non-fluorescent lactone or rearrange into lactam,they possess several major shortcomings. First, the disclosed secondaryamides are known to undergo hydrolysis under basic conditions(Boyarskiy, V. P. et al., Chem. Eur. J., 14:1784, 2008), resulting indissociation of a fluorescent dye from an analyte, so the dissociatedfluorescent dye will be detected by means of fluorescent spectroscopyand not the labeled biomolecule of interest. Second, conversion of acarboxylic acid into a secondary amide makes the dye molecule lesshydrophilic, which in turn increases the aggregation of labeledbiomolecules in aqueous media and increases the tendency for formingnon-fluorescent dye-dye dimers.

Therefore, there is a need for xanthene class fluorescent dyes that donot undergo formation of non-fluorescent lactone/lactam and, at the sametime, do not undergo hydrolysis in aqueous media.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention is directed to acarboxamide-substituted, lipophilic dye of the formula (I)

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently H or halogen,    -   R₃, R₄, R₇, and R₈ are independently H or C₁-C₅. Alternatively,        R₃ in combination with R₂ and/or R₈ in combination with R₉ form        a 5- or 6-membered saturated or unsaturated ring that is        optionally substituted with a C₁-C₅. Optionally, R₄ in        combination with R₅ and/or R₆ in combination with R₇ form a 5-        or 6-membered saturated ring that is optionally substituted with        a C₁-C₅ alkyl.    -   R₁₁ and R₁₂ are independently C₁-C₁₈ alkyl, branched or cyclic        saturated or unsaturated hydrocarbon group having up to 300        carbon atoms that is optionally interrupted by O or N atoms,        optionally further substituted with F, Cl, Br, I, a carboxylic        acid, a salt of carboxylic acid, or a carboxylic acid ester,    -   R₁₃ is a reactive group that is capable of modifying        biomolecules, and    -   R₁₄ is hydrogen or halogen.

In a second embodiment, the present invention is directed to ahydrophilic, carboxamide-substituted dye of the formula (I) in which:

-   -   R₁ through R₁₀, R₁₃, and R₁₄ are as described above,    -   R₁₁ and R₁₂ are independently polyethylene glycol having a        formula weight of 100-20000 Da or C₁-C₁₈ alkyl.

In a third embodiment, the present invention is directed to ahydrophilic, carboxamide-substituted dye of the formula (I) thatcontains at least one charged group in which:

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently H; halogen; SO₃X;        or SO₂NH—(CH₂)_(n)—SO₃X, where n=2-5, and X is H or a        counterion,    -   R₃, R₄, R₇, and R₈ are independently H or C₁-C₅ alkyl where each        alkyl is optionally further substituted with SO₃X.        Alternatively, R₃ in combination with R₂ and/or R₈ in        combination with R₉ form a 5- or 6-membered saturated or        unsaturated ring that is optionally substituted with a C₁-C₅        alkyl where each alkyl is optionally further substituted with        SO₃X. Optionally, R₄ in combination with R₅ and/or R₆ in        combination with R₇ form a 5- or 6-membered saturated ring that        is optionally substituted with a C₁-C₅ alkyl.    -   R₁₁ and R₁₂ are independently (CH₂)_(n)SO₃X, where X is H or a        counterion, and n=2-4, or a C₁-C₁₈ alkyl optionally further        substituted with a carboxylic acid, a salt of carboxylic acid,        SO₃X, or PO₃X,    -   R₁₃ is a reactive group, and    -   R₁₄ is hydrogen or halogen.

More specifically, R₁₁ and R₁₂ may be independently C₁-C₁₈ alkyl and(CH₂)_(n)—SO₃X where X is H or a counterion and n is 2, 3, or 4. R₁₁ andR₁₂ may be independently discrete or non-discrete polyethylene glycol.

In a fourth embodiment, the present invention is directed toheterobifunctional dyes of the general formula (I) where:

R₁ through R₁₀, R₁₃, and R₁₄ are as described above,

R₁₁ and R₁₂ may be independently a group consisting of orthogonalreactive pairs which undergo Staudinger ligation, copper-catalyzedHuisgen 1,3-dipolar cycloaddition, strain-promoted Huisgen 1,3dipolarcycloaddition, Inverse Electron Demand Diels-Alder cycloaddition, andhydrazone or oxime bond forming reactions.

In a fifth embodiment, the present invention is directed to acarboxamide-substituted dye of the formula:

in which:

-   -   R₁, R₂, R₉ and R₁₀ are independently hydrogen or halogen,    -   R₅ and R₆ are independently hydrogen, SO₃X, or        SO₂NH—(CH₂)_(n)—SO₃X, where X is H or a counterion, and n=2-5,

R₃, R₄, R₇, and R₈ are independently H or C_(i)-C₅ alkyl, where eachalkyl is optionally further substituted with halogen or SO₃X, where X isH or a counterion,

R₁₁ is C₁-C₁₈ alkyl, branched or cyclic saturated or unsaturatedhydrocarbon group having up to 300 carbon atoms that is optionallyinterrupted by O or N atoms, optionally further substituted with F, Cl,Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylicacid ester, SO₃X, or PO₃X, where X is H or a counterion,

-   -   R₁₂ is a reactive group, and    -   R₁₃ is hydrogen or halogen.

More specifically, R₁₁ may be discrete or non-discrete polyethyleneglycol. R₁₂ may be a biomolecule.

In a sixth embodiment, the present invention is directed to acarboxamide-substituted dye of the formula:

in which:

-   -   R₁, R₂, R₃, R₆, R₇, and R₈ are independently hydrogen, or C₁-C₅        alkyl, where each alkyl is optionally further substituted with        halogen or SO₃X, where X is H or a counterion,    -   R₉ and R₁₀ are independently hydrogen, SO₃X, or        SO₂NH—(CH₂)_(n)—SO₃X, where X is H or a counterion, and n=2-5,    -   R₄ and R₅ are independently hydrogen, —CH₃, or SO₃X, where X is        H or counterion,    -   R₁₁ is C₁-C₁₈ alkyl, branched or cyclic saturated or unsaturated        hydrocarbon group having up to 300 carbon atoms that is        optionally interrupted by O or N atoms, optionally further        substituted with F, Cl, Br, I, a carboxylic acid, a salt of        carboxylic acid, or a carboxylic acid ester, SO₃X, or PO₃X,        where X is H or a counterion,    -   R₁₂ is a reactive group, and    -   R₁₃ is hydrogen or halogen.

More specifically, R₁₁ may be discrete or non-discrete polyethyleneglycol. R₁₂ may be a biomolecule.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to one of ordinary skill in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will become more fully understood from thedetailed description given below and the accompanying drawing that isgiven by way of illustration only and is thus not limitative of thepresent invention.

FIG. 1 is a fluorescence emission spectra of IgG conjugates of Compound5 and Alexa Dye 546 at similar degrees of substitution and equal opticaldensities.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to theaccompanying drawing.

Unless defined otherwise, all terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents, patent applications, and publicationsreferred to throughout the disclosure herein are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those definitions in this sectionprevail.

The term “biomolecule” as used herein refers to a compound of biologicalorigin or of biological activity. Biomolecules include, for example, anucleic acid, a nucleotide, a protein, an amino acid, a carbohydratemonomer, and a polysaccharide. If the biomolecule is a nucleic acid, itmay be DNA, cDNA, RNA, or PNA and may comprise natural or unnaturalbases or internucleotide linkages, such as phosphodiesters,phosphorothioates, phosphoramidites, or peptide nucleic acids.

The term “reactive moiety” or “reactive group” herein refers to a moietythat can be coupled with another moiety without prior activation ortransformation. Some commercially sold molecules referred to herein aslinking moieties include those that react with free amines on the targetmolecule, such as N-hydroxysuccinimidyl, p-nitrophenyl,pentafluorophenyl and N-hydroxybenzotriazolyl ester, and those thatreact with free sulfhydryls present on the target molecule such asmaleimido, alpha-haloacetamido and pyridyldisulfides.

The term “linker” is a covalent linkage having 1-48 nonhydrogen atomsselected from the group consisting of C, N, O, P, and S and composed ofany combination of single, double, triple or aromatic carbon-carbonbonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, carbon-sulfur bonds, phosphorus-oxygen bonds, andphosphorus-nitrogen bonds. The non-cleavable linker is preferably (i) adivalent linear —(CH₂)_(x)— group or a —(CH₂CH₂O)_(x)— group wherein xis 1 to 25, (ii) a branched or cyclic alkane group, which is optionallysubstituted by at least one atom selected from the group consisting ofoxygen, substituted nitrogen, and sulfur, or (iii) absent. Thenon-cleavable linker is more preferably an alkyl having 1-6 carbon atomsor a discrete or non-discrete polyethylene glycol linker.

The term “ligand/receptor couple” as used herein refers to a pair ofmolecules having a substantially high affinity for binding specificallyto one another. One example of such a binding pair would be a cellreceptor and the ligand that binds that receptor. Another example wouldbe biotin and avidin, which are two molecules that have a strongaffinity for binding each other and having an association constant ofaround 10¹⁵. Other pairs include Peptide S and ribonuclease A,digoxigenin and its receptor and complementary oligonucleotide pairs.

Various methods exist which may be employed to bind the extended linkinggroup to a macromolecule or fragment. For example, to facilitate thisbinding, the extended linking group may be attached tobiomolecule-reactive groups, such as active ester groups, amino groups,sulfhydryl groups, carbohydrate groups, azido groups or carboxy groups.A variety of methodologies exist for reacting biomolecule-reactivegroups with macromolecules or macromolecule fragments. Examples of suchmethodologies are photo-crosslinking and glutaraldehyde crosslinkingStill other methods for affecting such coupling will occur to thoseskilled in the art. See, for examples of such methods: Hermanson, G. T.,Bioconjugate Techniques, Elsevier Science, London, 2008.

Active ester groups of the present invention may be selected such thatthey will not impair linkage of the extended linking group to a proteinor macromolecule. Those skilled in the art will appreciate that activeesters such as, for example, N-hydroxysuccinimide orN-hydroxysulfosuccinimide may be employed in the present invention.Alternatively, primary amino groups on the extended linking group may becoupled to primary amino groups on a protein by glutaraldehyde. Aminogroups on proteins may be coupled to carboxy groups on the extendedlinking group. In addition, the extended linking group may be modifiedwith a nitrophenylazide such that coupling to a protein will occur whenirradiated with visible light. Still other methods for affecting suchcoupling will occur to those skilled in the art.

The present invention describes xanthene-based, carboxamide-substituteddyes of the general formula (I) as well as the preparation, activation,and use of said dyes. The dyes of this invention possess a reactivegroup R₁₃ useful for preparation of fluorescent conjugates.

The compounds of this invention are rhodamines in which a carboxyl groupat the 2′ position is converted into secondary amide in order to preventformation of non-fluorescent lactone. The second carboxylic group at the5′ or 6′ position is optionally converted into a reactive group usefulfor preparing fluorescent conjugates.

One preferred embodiment of the present invention relates to lipophilicdyes of the general formula (I):

in which:

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently hydrogen or        halogen,    -   R₃, R₄, R₇, and R₉ are independently H or C₁-C₅ alkyl where each        alkyl is optionally further substituted with halogen,    -   R₁₁ and R₁₂ are independently a straight-chain, branched or        cyclic saturated or unsaturated hydrocarbon group having up to        30 carbon atoms that is optionally interrupted by O or N atoms,    -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

Exemplary reactive groups of R₁₃ are given in Table 1.

TABLE 1 Reactive Group Target Functional Group CarbodiimideAmine/Carboxyl Carbonyl Hydrazine Diazirine Nonselective HydrazideCarbohydrate (oxidized) Hydroxymethyl Phosphine Amine Imidoester AmineNHS-ester Amine PFP-ester Amine Psoralen Amine Pyridyl DisulfideSulfhydryl Vinyl Sulfone Sulfhydryl, amine, hydroxyl Terminal AlkyneAzide Azide Terminal alkyne, cyclooctyne Trans-Cyclooctene Tetrazine

To one skilled in the art, it will be apparent that there are multiplevariations of reactive groups useful for modifying a biomolecule withfluorescent dyes.

In yet another preferred embodiment of the present invention thelipophilic dyes has the general formula (II):

in which:

-   -   R₁, R₂, R₃, R₆, R₇, R₈, R₉, and R₁₀ are independently hydrogen        or C₁-C₅ alkyl,    -   R₄ and R₅ are independently hydrogen or CH₃,    -   R₁₁ and R₁₂ are independently a straight-chain, branched or        cyclic saturated or unsaturated hydrocarbon group having up to        30 carbon atoms that is optionally interrupted by O or N atoms,    -   R₁₃ is a reactive group, and    -   R₁₄ is hydrogen or halogen.

The lipophilic dyes of the general formula (I) and (II) are soluble innon-polar media and biological membranes and may be employed, forexample, for detecting membrane properties or for measuring moleculardistances.

In yet another embodiment, the present invention relates to hydrophilicdyes of the general formula (I) that contain a polyethylene glycol:

in which:

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently hydrogen, halogen,        or SO₂NH-R₁₅, where R₁₅ is polyethylene glycol having a formula        weight of 100-20000 Da,    -   R₃, R₄, R₇, and R₉ are independently H or C₁-C₅ alkyl,    -   R₁₁ and R₁₂ are independently polyethylene glycol having a        formula weight of 100-20000 Da or C₁-C₁₈ alkyl,    -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

In yet another preferred embodiment of the present invention, thehydrophilic dyes has the general formula (II):

in which:

-   -   R₁, R₂, R₃, R₆, R₇ and R₈ are independently hydrogen or C₁-C₃        alkyl,    -   R₄ and R₅ are independently hydrogen, CH₃, or CH₂—SO₂NH—R₁₅,        where R₁₅ is polyethylene glycol having a formula weight of        100-20000 Da,    -   R₉ and R₁₀ are independently hydrogen or SO₂NH—R₁₅, where R₁₅ is        polyethylene glycol having a formula weight of 100-20000 Da,    -   R₁₁ and R₁₂ are independently polyethylene glycol having a        formula weight of 100-20000 Da or C₁-C₁₈ alkyl,    -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

The hydrophilic dyes of the general formula (I) or (II) have goodsolubility in aqueous media and contain no negative charges. The use ofsuch hydrophilic dyes achieves a high degree of labeling ofbiomolecules, for example proteins, without aggregating/precipitatingthe biomolecules and without substantially changing the isoelectricpoint of the biomolecules. For example, IgG was labeled with Compound 4of the invention (see Table 2 below) with a very high degree ofsubstitution without precipitating the IgG. On the other hand, anattempt to achieve a similar degree of substitution using a structurallysimilar compound of US 2006/0154251A (Atto-Tec Dye 556) resulted incomplete precipitation of the IgG.

Surprisingly, it was possible to provide dyes, which functionalizeddifferently with various combinations of R₁₁ and R₁₂ according to thegeneral formula (I) and which have very good spectral properties, suchas the position of absorbance and fluorescence peaks, high extinctioncoefficient and high quantum yields. The disadvantage of lactone orlactam formation, which occurs with conventional dyes having acarboxylic acid at the 2′ position, is prevented by converting saidcarboxylic acid group into a secondary amide.

The spectral properties of dyes can be tuned by modifying the rhodaminecore of dyes. Several properties of such dyes are given in Table 2.

TABLE 2 Fluorescent dyes Abs/Em

518/539

531/551

556/573

556/573

546/562

601/623

Importantly, the introduction of the carboxamide group does notsubstantially alter spectral property of dyes, such as the position ofthe absorbance and fluorescence peaks, the high extinction coefficientand the high quantum yields. It was merely observed that the absorptionand emission maxima of some dyes red-shifted by 10 nm on average.

The dyes of this invention possess considerable advantages overcarboxamide dyes disclosed in WO 02/055512 and US 2006/0154251 A, wherethe carboxamide group at the 2′ position is used to connect the dyeswith biomolecules of interest. The carboxamide group at the 2′ positionis susceptible to hydrolysis under basic conditions (Boyarskiy, V. P. etal., Chem. Eur. J., 14:1784 2008). However, hydrolysis of carboxamidedyes, in which the carboxamide group at the 2′ position is used toconnect the dyes with biomolecules, results in dissociation of thefluorescent dye from an analyte. As such, the dissociated fluorescentdye will be detected by means of fluorescent spectroscopy rather thanthe labeled biomolecule of interest. The hydrolysis of carboxamide dyesof this invention will not result in dissociation of the dyes from thebiomolecule of interest. Rather, the hydrolysis of carboxamide dyes ofthis invention will only result in a slight change in spectralproperties of the conjugated dyes.

The properties of the dyes can be fine tuned by introducing variousmoieties at the amide group. Thus, it is possible, for example, toincrease the lipophilicity of the dyes by introducing a long alkyl chainas a moiety at the amide group. On the other hand, it is possible toincrease the hydrophilicity of the dyes by introducing a sugar residueor a long polyethylene glycol chain. Importantly, the dyes of thisinvention allow for the incorporation of a long polyethylene glycolchain without distancing the fluorescent dye from the conjugatedbiomolecule. Incorporation of a long polyethylene glycol chain intocarboxamide dyes is disclosed in WO 02/055512 and US 2006/0154251 A;however, the method used in these references possesses two majorshortcomings. First, it will result in substantial distancing of thefluorescent dye from the conjugated biomolecule, for example, when mPEG5000 or mPEG 10000 is used. Such distancing is very undesirable in someapplications. Second, it is synthetically challenging and impractical tomake such compounds.

In yet another embodiment, the present invention relates to hydrophilicdyes of the general formula (I) that possess at least one negativecharge.

in which:

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently hydrogen, halogen,        SO₃X, or SO₂NH—(CH₂)_(n)—SO₃X, where n=2-5, and X is H or a        counterion. Examples of suitable counterions include K⁺, Na⁺,        Cs⁺, Li⁺, Ca²⁺, Mg²⁺, ammonium, alkylammonium, alkoxyammonium        salts, or pyridinuim salts.    -   R₃, R₄, R₇, and R₈ are independently H or C₁-C₅ alkyl, where        each alkyl is optionally further substituted with SO₃X.    -   R₁₁ and R₁₂ are independently (CH₂)_(n)SO₃X, where X is H or a        counterion, and n=2-4, or a C₁-C₁₈ alkyl optionally further        substituted with a carboxylic acid, a salt of carboxylic acid,        SO₃X, or PO₃X,    -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

In yet another preferred embodiment of the present invention, thehydrophilic dyes has the general formula:

in which:

-   -   R₁, R₂, R₃, R₆, R₇ and R₈ are independently hydrogen, C₁-C₅        alkyl optionally further substituted with SO₃X, where n=2-5, and        X is H or a counterion. Examples of suitable counterions include        K⁺, Na⁺, Cs⁺, Ca²⁺, Mg²⁺, ammonium, alkylammonium,        alkoxyammonium salts, or pyridinuim salts.    -   R₄ and R₅ are independently hydrogen, CH₃, or CH₂SO₃X, where X        is H or counterion,    -   R₉ and R₁₀ are independently hydrogen or SO₃X, where X is H or        counterion,

R₁₁ and R₁₂ are independently (CH₂)_(n)SO₃X, and n=2-4, or a C₁-C₁₈alkyl optionally further substituted with a halogen, carboxylic acid, asalt of carboxylic acid, SO₃X, or PO₃X, where X is H or counterion,

-   -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

The fluorescence yield of protein-dye conjugates of the hydrophilic dyesof this invention are usually substantially higher than those ofstructurally and spectrally similar dyes having a carboxyl group at the2′ position of the “lower” aromatic ring. The enhanced fluorescence ispresumably a result of its inability to undergo formation intonon-fluorescent lactones and lactams. The comparison of fluorescenceyields of IgG labeled with Compound 5 and with structurally andspectrally similar Alexa Fluor 546 dye at a similar degree ofsubstitution and equal optical density revealed that Compound 5 hassubstantially increased resistance to quenching upon protein conjugationas shown in FIG. 1.

In still another aspect, the present invention relates toheterobifunctional dyes of the general formula (I)

in which:

-   -   R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently hydrogen, halogen,        SO₃X, or SO₂NH—(CH₂)_(n)SO₃X, where n =2-5, and X is H or a        counterion. Examples of suitable counterions include K⁺, Na⁺,        Cs⁺, Li⁺, Ca²⁺, Mg²⁺, ammonium, alkylammonium, alkoxyammonium        salts, or pyridinuim salts.    -   R₃, R₄, R₇, and R₈ are independently H or C₁-C₅ alkyl, where        each alkyl is optionally further substituted with SO₃X.

R₁₁ and R₁₂ are independently a reactive moiety partner of a pair oforthogonally reactive moieties that can react with each other in thepresence or absence of a catalyst without activation and both reactivemoieties are sufficiently stable under commonly applied biomoleculelabeling conditions or C₁-C₁₈ alkyl optionally further substituted witha carboxylic acid, a salt of carboxylic acid, SO₃X, or PO₃X,

-   -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

In yet another preferred embodiment of the present invention theheterobifunctional dyes has the general formula:

in which:

-   -   R₁, R₂, R₃, R₆, R₇ and R₈ are independently hydrogen, halogen,        SO₃X, or SO₂NH—(CH₂)_(n)SO₃X, where n=2-5, and X is H or a        counterion. Examples of suitable counterions include K⁺, Na⁺,        Cs⁺, Li⁺, Ca²⁺, Mg²⁺, ammonium, alkylammonium, alkoxyammonium        salts, or pyridinuim salts.    -   R₄ and R₅ are independently hydrogen, CH₃, or CH₂—SO₃X, where X        is H or counterion,    -   R₉ and R₁₀ are independently hydrogen or SO₃X, where X is H or        counterion,    -   R₁₁ and R₁₂ are independently a reactive moiety partner of a        pair of orthogonally reactive moieties that can react with each        other in the presence or absence of a catalyst without        activation and both reactive moieties are sufficiently stable        under commonly applied biomolecule labeling conditions or C₁-C₁₈        alkyl further substituted with a carboxylic acid, a salt of        carboxylic acid, SO₃X, or PO₃X,    -   R₁₃ is a reactive group or a biomolecule, and    -   R₁₄ is hydrogen or halogen.

Table 3 summarizes some of the preferred combinations of R₁₁/R₁₂ andR₁₃. These examples are not meant to be limiting but rather arerepresentative of some more useful functionalities used in manybiological applications.

TABLE 3 R₁₁/R₁₂ R₁₃ NHS-ester Trans-cyclooctene NHS-ester DBCO NHS-esterAzide NHS-ester Tetrazine

To one skilled in the art, it will be apparent that there are multiplevariations of reactive groups R₁₁/R₁₂ and R₁₃ useful for modifying abiomolecule with fluorescent dyes.

In still another aspect, the present invention relates to bio-orthogonalchemistry. The term bioorthogonal chemistry refers to any chemicalreaction that can occur in the presence of rich functionalities ofliving systems/biological media without interfering with nativebiochemical processes. In this strategy, one component of the conjugateis modified with a bioorthogonal functional group, while in a separatereaction, the other component is activated with a complementaryfunctional group of the bioorthogonal ligation pair. The twobioorthogonally-activated components are then mixed together andspontaneously react to form the specific conjugate. In certainembodiments, the bio-orthogonal reaction is a Cu-catalyzed version ofHuisgen 1,3-dipolar cycloaddition between an azide and terminal alkyne.In other embodiments, the reaction is carried out in the absence of sucha catalyst. Exemplary 1,3-dipole-functional compounds include, but arenot limited to, azides, nitrile oxides, nitrones, and diazo compounds.

In another aspect, the present invention relates to trans-cyclooctenesand tetrazines. The inverse-electron demand Diels-Alder cycloadditionreaction of trans-cyclooctenes (TCO) with tetrazines is a bioorthogonalreaction that possesses exceptional kinetics (k>800 M⁻¹ s⁻¹) andselectivity. Such excellent reaction rate constants are unparalleled byany other bioorthogonal reaction pair described to date. Exemplarydienophile compounds include, but are not limited to, norbornene andtrans-cyclooctenes.

By choosing appropriate click partners and fluorescent dyes, suchcompounds can be used for studying protein-protein interaction via FRETbetween dyes molecules.

In another embodiment of the present invention, acarboxamide-substituted dye has the following formula:

in which:

-   -   R₁, R₂, R₉ and R₁₀ are independently hydrogen or halogen,    -   R₅ and R₆ are independently hydrogen, SO₃X, or        SO₂NH—(CH₂)_(n)SO₃X, where X is H or a counterion, and n=2-5,    -   R₃, R₄, R₇, and R₈ are independently H or C₁-C₅ alkyl, where        each alkyl is optionally further substituted with halogen or        SO₃X, where X is H or a counterion,    -   R₁₁ is C₁-C₁₈ alkyl, branched or cyclic saturated or unsaturated        hydrocarbon group having up to 300 carbon atoms that is        optionally interrupted by O or N atoms, optionally further        substituted with F, Cl, Br, I, a carboxylic acid, a salt of        carboxylic acid, or a carboxylic acid ester, SO₃X, or PO₃X,        where X is H or a counterion,    -   R₁₂ is a reactive group, and    -   R₁₃ is hydrogen or halogen.

Preferably, R₁₁ may be discrete or non-discrete polyethylene glycol.Preferably, R₁₂ may be a biomolecule.

In yet another preferred embodiment, the present invention is directedto a carboxamide-substituted dye of the formula:

in which:

-   -   R₁, R₂, R₃, R₆, R₇, and R₈ are independently hydrogen, or C₁-C₅        alkyl, where each alkyl is optionally further substituted with        halogen or SO₃X, where X is H or a counterion,

R₉ and R₁₀ are independently hydrogen, SO₃X, or SO₂NH—(CH₂)_(n)SO₃X,where X is H or a counterion, and n=2-5,

R₄ and R₅ are independently hydrogen, CH₃, or SO₃X, where X is H orcounterion,

-   -   R₁₁ is C₁-C₁₈ alkyl, branched or cyclic saturated or unsaturated        hydrocarbon group having up to 300 carbon atoms that is        optionally interrupted by O or N atoms, optionally further        substituted with F, Cl, Br, I, a carboxylic acid, a salt of        carboxylic acid, or a carboxylic acid ester, SO₃X, or PO₃X,        where X is H or a counterion,    -   R₁₂ is a reactive group, and    -   R₁₃ is hydrogen or halogen.

Preferably, R₁₁ may be discrete or non-discrete polyethylene glycol.Preferably, R₁₂ may be a biomolecule.

One embodiment of the invention is a method of synthesis of carboxamidedyes according to the general formula (I) or (II) comprising thefollowing steps:

-   -   1. Condensation of the appropriate aminophenol with a methyl        ester of trimellitic anhydride in the presence or absence of        various acid catalysts or dehydrating agents. An aqueous workup,        usually followed by column chromatography, yields a mixture of        methyl esters of the desired dyes. The unsymmetrical xanthene        dyes can also be constructed in a stepwise fashion wherein a        selected aminophenol is condensed with a methyl ester of        trimellitic anhydride in a 1:1 ratio to yield a benzophenone,        which is optionally isolated, purified, and condensed with one        equivalent of a different aminophenol, yielding the asymmetric        dye.    -   2. Conversion of carboxylic acid at the 2′ position of the        “lower aromatic ring” into an activated ester with coupling        reagents followed by a reaction with a secondary amine in the        presence of non-nucleophilic bases, preferably at ambient        temperature. The preferred activating agent is HATU, and the        preferred non-nucleophilic base is triethylamine. Optionally,        the activated ester can be isolated.    -   3. Conversion of the methyl ester group of the carboxamide dyes        obtained in step 2 into a carboxylic acid. At this step, the        reaction should be very carefully controlled since a prolonged        reaction time will result in partial to full cleavage of the        secondary amide group.

Optionally, the carboxylic acid obtained in step 2 can be converted intoan activated ester or coupled to primary amines in the presence ofactivating agents well known in the art.

Yet another embodiment of the invention relates to the use of inventivecarboxamide dyes according to the general formula (I). The preparationof a dye conjugated using reactive dyes is well documented. See, forexamples of such methods: Hermanson, G. T., Bioconjugate Techniques,Elsevier Science, London, 2008. Conjugates typically result from mixingappropriate reactive dyes and the substance to be conjugated in asuitable solvent in which both are soluble.

For biological applications, the carboxamide dye of the invention istypically used in mostly aqueous media or aqueous-miscible solutionsprepared according to methods generally known in the art. The exactconcentration of the dye component depends on the experimentalconditions and the desired result, but typically ranges from about onenanomolar to one millimolar or more. The optimal concentration isdetermined experimentally. It is also preferred that the labeledantibody be purified by dialysis or by gel permeation chromatography toremove any unconjugated compounds. One of ordinary skill in the artwould know ways and means of purification.

In one aspect, biomolecules can be labeled according to the presentinvention by means of a kit. In certain instances, the kit comprises abuffer, a compound as disclosed in the instant application, purificationmedia, and the manual. Preferably, the kit contains a coupling buffersuch as 1 M KH₂PO₄ (pH 5), optionally with added acid or base to modifythe pH (e.g., pH 7.5 is preferred for reactions with succinimide estersand pH 6.5 is preferred for reactions with maleimides).

Conjugates having an ion-complexing moiety might be used as indicatorsfor calcium, sodium, zinc or other biologically important metal ions.Exemplary ion-complexing moieties are crown ethers, includingdiaryldiaza crown ethers (U.S. Pat. No. 5,405,975), and BAPTA chelators(U.S. Pat. No. 5,453,517).

While the invention has been described with references to a preferredembodiments, those skilled in the art will understand various changesmay be made and equivalents may be substituted for elements thereofwithout departing from the scope of the invention. In addition, manymodifications may be made to adapt a particular situation or materialsto the teaching of the invention without departing from the essentialscope thereof. Therefore, it is intended that the invention not to belimited to the particular embodiments disclosed but that the inventionwill include all embodiments falling within the scope of the appendedclaims. In this application, all units are in the metric system, and allamounts and percentages are by weight, unless otherwise expresslyindicated. Also, all citations referred to herein are expresslyincorporated herein by reference.

The following examples are offered to illustrate various embodiments ofthe invention but should not be viewed as limiting the scope of theinvention.

EXAMPLE S Example 1

A solution of 2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-7-ol (2 g,10.46 mmol) was heated to ca. 70° C. and cooled to room temperature.Methyl 1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxylate (2.156 g, 10.46mmol) was added, and the reaction mixture was brought to reflux. Thereaction mixture was refluxed overnight. According to TLC analysis, onemajor, non-fluorescent product was formed, and a small amount of highlyfluorescent compound was also detected by TLC. The reaction mixture wasconcentrated under reduced pressure, DMF (25 mL) was added,2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-7-ol (2 g, 10.46 mmol) andtrimethylsilylpolyphosphate (dehydrating agent, 12 mL) were added, andthe reaction mixture was refluxed for ca. 60 min. Upon completion, thereaction mixture was cooled to room temperature and concentrated underreduced pressure to provide an oily compound that became semi-solid uponstanding at room temperature. The crude product (ca. 3 g) was purifiedon silica gel (DCM:MeOH 10:1) to provide 1.35 g of Compound 7 as a redsolid.

Example 2

HATU (2.91 g, 7.64 mmol) was added to a solution of Compound 7 (3.25 g,5.88 mmol) and N,N-diethylpropan-2-amine (2.033 g, 17.64 mmol) in DMF(50 ml) at room temperature, and the reaction mixture was stirred forca. 30 min at room temperature. A suspension of2-(methylamino)ethanesulfonic acid (0.982 g, 7.06 mmol) and DIEA (ca. 2mL) was added to the reaction mixture, and the reaction mixture wasstirred overnight at room temperature. According to TLC analysis, all ofthe substrate was consumed, and one product was detected by TLC. Thereaction mixture was concentrated under reduced pressure to provide anoily compound. The crude product was purified on silica gel (DCM:MeOH10:1 to 3:1) to provide 3.7 g of Compound 8 as a red solid.

Example 3

A solution of lithium hydroxide (0.960 g, 27.4 mmol) in water (7.50 ml)was added to a solution of Compound 8 (3.7 g, 5.48 mmol) in MeOH (15ml), and the reaction mixture was stirred for ca. 60 min. According toTLC analysis, all of the substrate was consumed, and a small amount ofdiacid was formed by hydrolysis of the amide group. Prolonged reactiontime usually results in substantial formation of the diacid. Thereaction mixture was concentrated under reduced pressure to providecrude 9 as a red solid. The crude product was purified on silica gel(DCM:MeOH 10:1 to 3:1 +0.5% AcOH) to provide 1.4 g of Compound 9 as ared solid.

Example 4

Compound 9 (1.21 g) was added in several portions to 30% fuming sulfuricacid at −10° C. over 2 hours, the reaction mixture was brought to 4° C.,and stirred overnight. According to HPLC, all of the substrate wasconsumed, and one product (two isomers) was formed. The reaction mixturewas carefully poured into ice (cooled to ca. −50° C.) and continuedcooling in a dry ice-acetone bath. Upon addition, the reaction mixturewas slowly warmed to room temperature, loaded onto a column packed withC18 silica gel, and chromatographed (water to 30% MeOH in water,gradient). Fractions containing the product were pooled and concentratedunder reduced pressure to provide 0.45 g of Compound 5.

Example 5

DCC (0.102 g, 0.493 mmol) was added to a solution of Compound 5 (0.3 g)and NHS (0.048 g, 0.42 mmol) in DMF (20 mL) at room temperature, and thereaction mixture was stirred overnight. According to HPLC, all of theacid was converted into NHS ester. The reaction mixture was placed intoa refrigerator for 24 hours, and the precipitate was filtered. Thereaction mixture was poured into EtOAc (150 mL) and stirred for ca. anhour. The precipitate was filtered, washed with EtOAc, and dried on anoil pump to provide 320 mg of Compound 10.

Example 6

HATU (2.16 g, 5.66 mmol) was added to a solution of Compound 7 (2.41 g,4.36 mmol) and N,N-diethylpropan-2-amine (1.5 mL) in DMF (30 ml) at roomtemperature, and the reaction mixture was stirred for ca. 30 min at roomtemperature. A solution of ethyl-mPEG11 amine (3.9 g) was added to thereaction mixture, and the reaction mixture was stirred overnight at roomtemperature. According to TLC analysis, all of the substrate wasconsumed, and one product was detected by TLC. The reaction mixture wasconcentrated under reduced pressure to provide an oily compound. Thecrude product was purified on silica gel (DCM:MeOH 15:1 to 10:1) toprovide 2.1 g of Compound 11 as a red solid.

Example 7

A solution of lithium hydroxide (0.686 g, 19.6 mmol) in water (5 ml) wasadded to a solution of Compound 11 (2.0 g) in MeOH (10 ml), and thereaction mixture was stirred for ca. 60 min. According to TLC analysis,all of the substrate was consumed, and some amount of diacid was formed.Prolonged reaction time usually results in substantial formation of thediacid. The reaction mixture was concentrated under reduced pressure toprovide crude 12 as a red solid. The crude product was purified onsilica gel (DCM:MeOH 15:1 to 5:1 +0.5% AcOH) to provide 0.78 g ofCompound 12 as a red solid.

Example 8

DCC (0.5 g, 2.49 mmol) was added to a solution of Compound 12 (0.78 g)and NHS (0.2 g, 2.10 mmol) in DMF (10 mL) at room temperature, and thereaction mixture was stirred overnight. According to HPLC, all of theacid was converted into NHS ester. The reaction mixture was placed intoa refrigerator for 24 hours, and the precipitate was filtered. Thereaction mixture was concentrated under reduced pressure andchromatographed on silica gel (DCM:MeOH 10:1 to 5:1+0.5% AcOH) toprovide 436 mg of Compound 13.

Example 9

A solution of 3-azidopropan-1-amine (2 g, 19.98 mmol) and1,2-oxathiolane 2,2-dioxide (2.440 g, 19.98 mmol) in DCM (10 mL) wasstirred overnight at room temperature. A white precipitate was filteredand washed with a small amount of THF-Et₂O and dried on an oil pump toprovide 2.35 g of 3-((3-azidopropyl)amino)propane-1-sulfonic acid.

Example 10

HATU (0.894 g, 2.352 mmol) was added to a solution of Compound 7 (1 g,1.809 mmol) and N,N-diethylpropan-2-amine (0.625 g, 5.43 mmol) in DMF(15 ml) at room temperature, and the reaction mixture was stirred forca. 30 min. A suspension of 3-((3-azidopropyl)amino)propane-1-sulfonicacid (0.422 g, 1.900 mmol) and DIEA (ca. 0.5 mL) was added, and thereaction mixture was stirred overnight at room temperature. According toTLC analysis, all of the substrate was converted into product. Thereaction mixture was concentrated under reduced pressure andchromatographed on silica gel (DCM:MeOH 20:1 to 10:1 to 5:1) to provide0.61 g of Compound 14.

Example 11

Compound 14 was converted into Compound 15 in the same way that Compound11 was converted into Compound 13.

Example 12

A solution of DBCO-Amine (4 g, 14.48 mmol) (Click Chemistry Tools,Scottsdale, Ariz.) and 1,2-oxathiolane 2,2-dioxide (1.591 g, 13.03 mmol)in DCM (20 mL) was stirred overnight at room temperature. A whiteprecipitate was filtered, washed with a small amount of THF-Et₂O, anddried on an oil pump to provide 3.53 g (8.86 mmol, 61%) of Compound 16.

Example 13

HATU (0.395 g, 1.039 mmol) was added to a solution of Compound 7 (0.5 g,0.903 mmol) and N,N-diethylpropan-2-amine (0.312 g, 2.71 mmol) in DMF(10 ml) at room temperature, and the reaction mixture was stirred forca. 30 min. A suspension of Compound 16 (0.360 g, 0.903 mmol) and DIEA(ca. 0.3 mL) was added, and the reaction mixture was stirred overnightat room temperature. According to TLC analysis, all of the substrate wasconverted into product. The reaction mixture was concentrated underreduced pressure and chromatographed on silica gel (DCM:MeOH 20:1 to10:1 to 5:1) to provide 0.60 g (0.624 mmol, 71%) of Compound 17.

Example 14

Compound 17 was converted into Compound 18 in the same way that Compound11 was converted into Compound 13.

Compounds 19 and 20 were prepared from commercially available TCO-Amineand tetrazine-amine using the procedure described in Examples 12, 13,and 14.

Compound 1, 2, and 6 were prepared in the same way as dyes 3, 4, or 5using corresponding aminophenols. The corresponding aminophenols wereprepared according to syntheses known from the literature or processesknown to the skilled person.

Example 15

Compound 21 was prepared according to Examples 6, 7, and 8.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

1. A carboxamide-substituted dye of the formula (I)

in which: R₁, R₂, R₅, R₆, R₉ and R₁₀ are independently hydrogen,halogen, SO₃X, or SO₂NH—(CH₂)_(n)SO₃X, where X is H or a counterion, andn=2-5, R₃, R₄, R₇, and R_(g) are independently H or C₁-C₅ alkyl, whereeach alkyl is optionally further substituted with halogen or SO₃X, whereX is H or a counterion, or R₃ in combination with R₂ and/or R₈ incombination with R₉ form a 5- or 6-membered saturated or unsaturatedring that is optionally substituted with a C₁-C₅ alkyl, where each alkylis optionally further substituted with SO₃X, or R₄ in combination withR₅ and/or R₆ in combination with R₇ form a 5- or 6-membered saturatedring that is optionally substituted with a C₁-C₅ alkyl, R₁₁ and R₁₂ areeach independently a straight-chain, branched or cyclic saturated orunsaturated hydrocarbon group having up to 300 carbon atoms that isoptionally interrupted by O or N atoms, optionally further substitutedwith F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or acarboxylic acid ester, SO₃X, or PO₃X, where X is H or a counterion, R₁₃is a reactive group, and R₁₄ is hydrogen or halogen.
 2. The compoundaccording to claim 1, wherein R₁₁ and R₁₂ are independently C₁-C₁₈ alkyland (CH₂)_(n)SO₃X where X is H or a counterion and n is 2, 3, or
 4. 3.The compound according to claim 1, wherein R₁₁ and R₁₂ are independentlydiscrete or non-discrete polyethylene glycol.
 4. The compound accordingto claim 1, wherein either R₁₁ or R₁₂ is independently selected from thegroup consisting of orthogonal reactive pairs which undergo Staudingerligation, copper-catalyzed Huisgen 1,3-dipolar cycloaddition,strain-promoted Huisgen 1,3dipolar cycloaddition, Inverse ElectronDemand Diels-Alder cycloaddition, and hydrazone or oxime bond formingreactions.
 5. The compound according to any one of claims 1 to 4,wherein R₁₃ is a biomolecule.
 6. A carboxamide-substituted dye of theformula:

in which: R₁, R₂, R₉ and R₁₀ are independently hydrogen or halogen, R₅and R₆ are independently hydrogen, SO₃X, or SO₂NH—(CH₂)_(n)SO₃X, where Xis H or a counterion, and n=2-5, R₃, R₄, R₇, and R₈ are independently Hor C₁-C₅ alkyl, where each alkyl is optionally further substituted withhalogen or SO₃X, where X is H or a counterion, R₁₁ is C₁-C₁₈ alkyl,branched or cyclic saturated or unsaturated hydrocarbon group having upto 300 carbon atoms that is optionally interrupted by O or N atoms,optionally further substituted with F, Cl, Br, I, a carboxylic acid, asalt of carboxylic acid, or a carboxylic acid ester, SO₃X, or PO₃X,where X is H or a counterion, R₁₂ is a reactive group, and R₁₃ ishydrogen or halogen.
 7. The compound according to claim 6, wherein R₁₁is discrete or non-discrete polyethylene glycol.
 8. The compoundaccording to claim 6 or claim 7, wherein R₁₂ is a biomolecule.
 9. Acarboxamide-substituted dye of the formula:

in which: R₁, R₂, R₃, R₆, R₇, and R₈ are independently hydrogen, orC₁-C₅ alkyl, where each alkyl is optionally further substituted withhalogen or SO₃X, where X is H or a counterion, R₉ and R₁₀ areindependently hydrogen, SO₃X, or SO₂NH—(CH₂)_(n)SO₃X, where X is H or acounterion, and n=2-5, R₄ and R₅ are independently hydrogen, CH₃, orSO₃X, where X is H or counterion, R₁₁ is C₁-C₁₈ alkyl, branched orcyclic saturated or unsaturated hydrocarbon group having up to 300carbon atoms that is optionally interrupted by O or N atoms, optionallyfurther substituted with F, Cl, Br, I, a carboxylic acid, a salt ofcarboxylic acid, or a carboxylic acid ester, SO₃X, or PO₃X, where X is Hor a counterion, R₁₂ is a reactive group, and R₁₃ is hydrogen orhalogen.
 10. The compound according to claim 9, wherein R₁₁ is discreteor non-discrete polyethylene glycol.
 11. The compound according to claim9 or claim 10, wherein R₁₂ is a biomolecule.